Specific Aim 1. Regulation of Synaptic Vesicle Dynamics and Release. Information coding in the brain depends on the timing of action potentials, which is influenced by integration of unitary excitatory inputs. The size and shape of excitatory postsynaptic currents (EPSCs) are two decisive factors in tuning the temporal and spatial precision of spiking and can be modulated by the SV fusion process. Our recent studies using the snapin-deficient cortical neurons combined with gene rescue experiments revealed a crucial role for Snapin in enhancing the efficacy of SV priming and in fine-tuning the precision of synchronous release (Pan et al., Neuron 2009). Snapin mutant neurons exhibit EPSCs with multiple peaks and fail to follow sustained firing under high-frequency stimulation. Re-introducing snapin into the mutant presynaptic neurons effectively accelerates EPSC kinetics by boosting the synchronicity of SV fusion. Thus, our studies reveal the role of Snapin as a unique synchronizer of SV fusion at central synapses. Several groups have independently reported an interaction between Snapin and dysbindin (BTNBP1)the product of a susceptibility gene found among the common genetic variations associated with schizophrenia. Our recent study also identified that Snapin acts as dynein motor adaptor for late endosomes (Cai et al., Neuron 2010). This raises an interesting question whether Snapin regulates synaptic vesicle transport and dynamics at release sites, thus contributing to regulation of synaptic transmission. Our current studies aim at (1) elucidating mechanisms by which Snapin regulates synchronized synaptic transmission;(2) determining whether Snapin-dynein transport complex regulates synaptic vesicle density and dynamics at presynaptic terminals;and (3) evaluating Snapins role in the cognitive impairment prominent in schizophrenia. Specific Aim 2. Regulation of Presynaptic Cargo Transport and its Impact on Synaptic Plasticity. The formation of new synapses and remodeling of existing synapses play an important role in the various forms of synaptic plasticity and require the targeted delivery of newly synthesized synaptic components from the trans-Golgi network (TGN) in the soma to the synaptic terminals. Thus, efficient axonal transport of these newly synthesized components to nascent presynaptic boutons is critical in response to neuronal activity. Substantial evidence suggests that AZ precursor carriers are generated from TGN and traverse the developing axon to nascent synapses. Cargo vesicles must attach to their transport motors with a high degree of specificity to preserve cargo identity and targeted trafficking. However, the molecular identities of the motor-adaptor complex essential for assembling presynaptic terminals in developing neurons and in remodeling synapses of mature neurons in response to neuronal activity remain unknown. Our previous studies established that syntabulin is a motor adaptor capable of joining KIF5B and syntaxin-1 and enables syntaxin-1 transport to neuronal processes (Su et al., Nature Cell Biology, 2004). Using time-lapse imaging in live hippocampal neurons, we further demonstrate that the transport complex of syntaxin-1-syntabulin-KIF5B mediates axonal transport of the AZ components essential for presynaptic assembly. Syntabulin loss-of-function blocks formation of new presynaptic boutons during activity-dependent synaptic plasticity in developing neurons (Cai et al., J Neuroscience 2007). Our studies establish that kinesin-mediated and MT-based anterograde axonal transport is another critical factor in the cellular mechanism underlying activity-dependent presynaptic plasticity. Our recent study further demonstrated the critical role of syntabulin-mediated axonal transport in the maintenance of presynaptic function and regulation of synaptic plasticity in well-matured sympathetic SCG neurons in culture (Ma et al., J Neuroscience 2009). Our findings provide a molecular basis for future studies. Conditional syntabulin knockout mice are in generation and we will use this genetic mouse line to (1) determine whether deficiency in syntabulin/KIF5-mediated anterograde axonal transport has any impact on synapse formation and maintenance, and synaptic plasticity;(2) determine whether the motor-adaptor complex regulates the transport rate in response to synaptic activity;(3) identify the sorting signals for the axon-targeted delivery of the AZ cargo. Specific Aim 3. Retrograde Transport and Impact on Neuronal Autophagy-Lysosomal Function. Maintaining cellular homeostasis in neurons depends on efficient intracellular transport. Late endocytic trafficking, which delivers target materials into lysosomes, is critical for maintaining efficient degradation capacities via autophagy-lysosomal pathways. Autophagy-lysosomal system is essential for quality control of intracellular components and mitochondria, and maintenance of cellular homeostasis. An impaired autophagy-lysosomal system has been associated with the pathogenesis of several major neurodegenerative diseases. However, the mechanisms regulating the autophagy-lysosomal system in neurons remain incompletely understood. Dynein-mediated retrograde transport can enhance late endocytic trafficking to some, where lysosomes are predominantly localized, and drive late endosomes and lysosomes close enough to fuse with higher efficiency, thus ensuring proper autophagy-lysosomal function. In addition to its association with SVs, Snapin is present in cytosol and membrane-associated fractions in neuronal and non-neuronal cells and is co-purified with late endocytic organelles. Our recent study uncovered a critical role for Snapin in regulating late endocytic transport and membrane trafficking (Cai et al., Neuron 2010). Snapin acts as a motor adaptor by attaching dynein to late endosomes. Snapin (-/-) neurons exhibit aberrant accumulation of immature lysosomes, impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene. Thus, our study highlights new mechanistic insights into how Snapin-dynein coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function. Our research goal is to identify the cellular pathways for clearance of aggregation-prone proteins by regulating the autophagy-lysosomal system. The snapin KO mouse provides us with a unique genetic tool for characterizing the role of late endocytic transport in neurodegeneration. The conditional snapin KO mice are generated and are being crossing with several disease mouse lines including mutant SOD1-linked ALS disease model. These studies will provide genetic evidence as to whether manipulating the late endocytic pathway will ultimately lead to new therapeutic approaches. Pursuing these investigations will advance our knowledge of fundamental processes that may affect human neurological disorders and is thus the very essence of the mission of the National Institute of Neurological Disorders and Stroke. Related publications from the lab: Qingning Su, Qian Cai, Claudia Gerwin, Carolyn L. Smith, Zu-Hang Sheng (2004) Syntabulin: a microtubule-associated protein implicated in syntaxin transport in neurons, Nature Cell Biology 6, 941-953. Qian Cai, Pingyue Pan, and Zu-Hang Sheng. (2007). Syntabulin-kinesin-1 family 5B-mediated axonal transport contributes to activity-dependent presynaptic assembly. Journal of Neuroscience 27, 7284-7296. Ping-Yue Pan, Jin-Hua Tian and Zu-Hang Sheng (2009). Snapin Facilitates the Synchronization of Synaptic Vesicle Fusion. Neuron 61, 412-424.